To understand how color information is processed in the fly brain, we set out to identify the second/third-order interneurons in the medulla ganglion, which synapse with R7 (UV channel), R8 (green/blue channel), or lamina neurons (green channel). To this end, we exploited the fact that the photoreceptor neurons (but not the other neurons in the visual system) use histamine as their neurotransmitter and, therefore, their synaptic target neurons must express histamine-gated chloride channels (encoded by the ort gene). We constructed a transgenic fly line that expresses the Gal4 transcription factor under the control of the ort promoter. Using the ort-Gal4 line to drive a membrane-tethered GFP marker, we identified subsets of medulla and lamina neurons that likely serve as synaptic targets for R7/8 and R1-6, respectively. Electron-microscopic study confirmed that these ort (+) medulla neurons indeed form synaptic connections with R7 and R8 axonal termini. Furthermore, expressing the ort gene under the control of ort-Gal4 completely rescues the electrophysiology and behavioral defects of the ort mutants, indicating that our ort-Gal4 construct faithfully recapitulates the endogenous ort expression pattern.[unreadable] [unreadable] To determine the information flow in the visual system, we identified (a) the connection patterns of these ort(+) interneurons in the medulla, (b) their dendritic and axonal compartments, and (c) their expression of neurotransmitters and receptors. Using a number of enhancer-trap lines, we found that ort (+) neurons can be further categorized into three subtypes (cholinergic, glutaminergic, and GABAnergic) according to the neurotransmitters they use. To determine the connection patterns of these R7/R8 target neurons, we performed single-cell mosaic analyses using a flipase-based genetic system. We identified seven types of first-order interneurons, including one medulla intrinsic cell type, which communicates between the external and internal medulla neuropils, and six types of transmedullary neurons (projection neurons). The projection neurons extend dendritic arbors in various medulla layers and project axons to the lobula neuropil to form a topographic map. Two types of projection neurons appear to receive input from two color-channels, suggesting that they might function as color-opponent or summation neurons. The Tm5 type neurons extend dendritic arbors in the M2 (L2) and M3 (R8) layers and convey both green and blue color information to the higher visual center, the lobula. Similarly, the Tm20 type neurons connect both R7 (UV channel) and R8 (blue) inputs to a specific layer in the lobula. We propose that the Tm5 and Tm20 neurons are color-opposing neurons, which calculate the intensity differences between spectra. Our study suggests that fly color-vision circuit shares a similar design with those of primates. Furthermore, these results highlight the lobula ganglion as the higher visual center for color vision.